Cardiac Calcium Channels Research Articles

Impact of Dihydrotestosterone on L-Type Calcium Channels in Human Ventricular Cardiomyocytes

Objectives. Reports of testosterone effects on cardiovascular morbidity remain contradictory. Besides modulating cardiovascular risk factors recent evidence indicates direct actions of testosterone on cardiac tissue. However, the impact on human cardiac L-type calcium channels that play a central role in electro-mechanical coupling is unknown. Methods and Results. Human ventricular myocytes were isolated from patients undergoing heart transplantation. Patch-clamp experiments in whole-cell configuration were performed to evaluate the effect of dihydrotestosterone on cardiac L-type calcium current ICa,L. Treatment of cultured cardiomyocytes with dihydrotestosterone 100 nmol/L for 24–30 h increased the whole-cell ICa,L current density from 2.32 ± 0.17 pA/pF (n = 11) to 3.21 ± 0.17 pA/pF (n = 14) at +10 mV (p = 0.01) without shifting the current–voltage relation. This effect was associated with a 1.35-fold higher expression of the pore-forming CaV1.2 (α1c) subunit of L-type calcium channels in dihydrotestosterone-treated myocytes compared with controls (p = 0.03). Conclusions. Dihydrotestosterone treatment increased L-type calcium current density by the upregulation of CaV1.2 in human ventricular myocytes. These data provide a possible explanation for dihydrotestosterone effects on the cardiovascular system in androgenic steroid abuse.

Abstract 1875: L-type Calcium Channel C-terminus Suppresses L-type Calcium Channel Transcription in Cardiomyocytes

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. In this study we test the hypothesis that a mobile carboxyl-terminal cleavage product (CCt) of the cardiac L-type calcium channel (Ca V 1.2) auto-regulates expression. Our ongoing in vivo studies showed that persistent Ca V 1.2 block induced an up-regulation of Ca V 1.2 mRNA, protein and I Ca,L . In the present study we test the hypothesis that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We created eGFP-CCt fusion constructs to explore CCt regulation of the Ca V 1.2 promoter, and to report sub-cellular localization of CCt in cardiac myocytes. Developing cardiac myocytes were cultured in 10% FBS. Over-expression of native, full-length CCt causes a 34 ± 8% decrease of Ca V 1.2 promoter activity, and truncated CCt caused 80 ± 3% decrease (n = 12). Wild-type CCt distributes into cytosol and nucleus, with a greater presence in the nucleus than cytosol. A deletion mutant of CCt was also tested. Truncated CCt has a greater relative affinity for the nucleus than wild-type CCt. This is consistent with increased suppression of Ca V 1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation (ChIP) assay showed that CCt interacts with the Ca V 1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing NKX2.5/MEF2, C/EBp, and a cis regulatory module identified in silico . We next explored whether fetal cardiac myocyte Ca V 1.2 was regulated by serum in vitro . We tested ANF promoter activity as a positive control, and measured serum-response, versus serum-withdrawal on Ca V 1.2 promoter, mRNA, protein, and L-type current (I Ca,L ) from fetal mouse ventricular myocytes. Serum increased ANF promoter activity and cell size. Serum withdrawal increased Ca V 1.2 promoter activity, Ca V 1.2 protein, and I Ca,L . Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analysis, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca V 1.2 auto-regulates Ca V 1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca V 1.2 links Ca handling to nuclear signaling.

Abstract 5309: beta-Arrestin 1 (bArr1) and Ankyrin 2 (ANK2) Regulate Cav1.2 Trafficking through Complex G Protein Mediated Signaling

Mechanisms governing the trafficking of cardiac voltage dependent calcium channels (Ca v 1.2) remain largely unknown. We have previously shown that neuronal calcium channels are internalized into clathrin coated vesicles upon G protein coupled receptor (GPCR) activation. Here, we hypothesize that GPCR mediated signaling regulate Ca v 1.2 trafficking and surface expression through interaction with cytoskeletal and signaling molecules. Methods : Sequential immunoprecipitation in adult rat cardiomyocytes was used to investigate the interaction between Ca v 1.2, cytoskeletal and signaling molecules. Live cell imaging and immunofluorescence microscopy were used to measure changes in the cellular localization of Ca v 1.2 and these proteins upon activation of GPCR signaling. Results : Cav1.2 interacts with ANK2, βArr1 and spectrin. Sustained β adrenergic receptor (βAR) activation increases Cav1.2/spectrin and decreases Ca v 1.2/ANK2 and Ca v 1.2/βArr1 interactions. Sustained βAR activation induces rapid (<5 min) Cav1.2 internalization and dissociation from ANK2 as Ca v 1.2/ANK2 co-localization is markedly reduced (−58% p<0.01) in isoproterenol treated myocytes. A cell permeant peptide (1.4 μg/ml) that disrupts Ca v 1.2/ANK2 interaction decreases (−40±12%, p<0.01) Cav1.2 cell surface expression. Pretreatment with pertussis toxin prevents Ca v 1.2 internalization suggesting that G i/o mediates this response. Similarly, Src kinase inhibition reduces (by 90%) Ca v 1.2 internalization. Differential labeling of pre-existing Ca v 1.2 at the cell membrane and newly inserted Ca v 1.2 using fluorophore conjugated dihydropyridines reveal that activation of G i/o proteins underlies Ca v 1.2 internalization and insertion. In contrast, activation of the muscarinic M2 receptor (10μM carbachol) causes only insertion of new channels (n=14) and fails to alter the association of Ca v 1.2 with ANK2 and spectrin. Finally PI3 kinase inhibition (10nM wortmannin) prevents Ca v 1.2 membrane insertion. Conclusions : Our findings reveal novel signaling mechanisms that regulate Ca v 1.2 trafficking, internalization and membrane insertion in a receptor subtype dependent manner. These mechanisms may be central to pathologies associated with a hyperadrenergic state. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Atrial Glutathione Content, Calcium Current, and Contractility

Atrial fibrillation (AF) is characterized by decreased L-type calcium current (I(Ca,L)) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and I(Ca,L) in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in I(Ca,L). To test the hypothesis that glutathione levels were mechanistically linked with the reduction in I(Ca,L), dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of I(Ca,L) in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored I(Ca,L) to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of I(Ca), the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of I(Ca,L) observed in AF.

The Tumor Suppressor eIF3e Mediates Calcium-Dependent Internalization of the L-Type Calcium Channel CaV1.2

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

A simulation study of voltage-dependent inactivation in the cardiac L-type calcium channel

A simulation study of voltage-dependent inactivation in the cardiac L-type calcium channel

Issue Highlights

HomeCirculationVol. 115, No. 4Issue Highlights Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIssue Highlights Originally published30 Jan 2007https://doi.org/10.1161/circ.115.4.427Circulation. 2007;115:427LOSS-OF-FUNCTION MUTATIONS IN THE CARDIAC CALCIUM CHANNEL UNDERLIE A NEW CLINICAL ENTITY CHARACTERIZED BY ST-SEGMENT ELEVATION, SHORT QT INTERVALS, AND SUDDEN CARDIAC DEATH, by Antzelevitch et al.There are multiple causes of inherited arrhythmia syndromes, many due to ion channelopathies. Mutations in ion channel encoding genes lead to clinical phenotypes of arrhythmias, conduction abnormalities, and vulnerability to sudden death. Recently, rather than typical long- or short-QT syndrome, Brugada syndrome, or conduction defects, reports of several overlapping clinical features bridge the spectrum of inherited arrhythmias. In this issue of Circulation, Antzelevitch and colleagues report finding mutations in L-type calcium channel encoding genes in 7/82 Brugada syndrome patients without SCN5A sodium channel or known long-QT syndrome mutations. Of these, 3 had an interesting clinical constellation of right precordial ST-segment elevation (resembling Brugada syndrome), short QT intervals (resembling short-QT syndrome), and sudden cardiac death. They characterize the clinical, genetic, and family histories and perform detailed in vitro biophysical functional analysis of the resultant calcium channel mutations, demonstrating loss of function deficits in this hybrid inherited arrhythmia disorder. See p 442.TRIGLYCERIDES AND THE RISK OF CORONARY HEART DISEASE: 10 158 INCIDENT CASES AMONG 262 525 PARTICIPANTS IN 29 WESTERN PROSPECTIVE STUDIES, by Sarwar et al.Although there have been many previous reports on the importance of serum triglyceride concentrations and the risk of coronary heart disease, the strength of the association and the existence of sex differences have remained controversial. Sarwar and colleagues report on case-control studies from Reykjavik and EPIC-Norfolk studies and present an updated meta-analysis in almost 300 000 participants in 29 Western prospective studies. They observed that the long-term stability of triglyceride measurements over time was similar to blood pressure and cholesterol values, and they account for regression dilution bias in their analysis. The authors observed that the top third of log-triglyceride values was associated with a 50% increased risk of coronary heart disease compared with the lower third. They also noted no major differences in risk by sex or in studies conducted among fasting versus nonfasting participants. The investigators note, however, that their data cannot resolve whether triglycerides per se are causally responsible for the excess coronary heart disease risk. See p 450.PROGNOSTIC VALUE OF HEART RATE INCREASE AT ONSET OF EXERCISE TESTING, by Leeper et al.Non–ST segment variables derived from the exercise ECG test have recently been shown to provide important prognostic information and, accordingly, have been the focus of much attention. Peak exercise capacity, the difference in heart rate from rest to peak exercise, and the fall in heart rate during early recovery can be easily measured during routine exercise testing and thus can be readily applied in clinical practice. In this issue, Leeper et al evaluate the prognostic value of heart rate responses at the start of exercise in 1959 patients referred for clinical treadmill testing and compare it to that provided by other variables derived from the same test. In an accompanying editorial, Bernard Chaitman offers important and practical insights regarding these interesting findings. See p 468 (editorial on p 430).Visit http://circ.ahajournals.org:Images in Cardiovascular MedicineCardiac Leiomyosarcoma. See p e47. Download figureDownload PowerPointKussmaul’s Sign in Right Ventricular Dysfunction. See p e49.Hemodynamic Consequences of Massive Coronary Air Embolism. See p e51.CorrespondenceSee p e54. Previous Back to top Next FiguresReferencesRelatedDetails January 30, 2007Vol 115, Issue 4 Advertisement Article InformationMetrics https://doi.org/10.1161/circ.115.4.427 Originally publishedJanuary 30, 2007 PDF download Advertisement

Loss-of-Function Mutations in the Cardiac Calcium Channel Underlie a New Clinical Entity Characterized by ST-Segment Elevation, Short QT Intervals, and Sudden Cardiac Death

Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity that consists of an ST-segment elevation in the right precordial ECG leads, a shorter-than-normal QT interval, and a history of sudden cardiac death. Eighty-two consecutive probands with Brugada syndrome were screened for ion channel gene mutations with direct sequencing. Site-directed mutagenesis was performed, and CHO-K1 cells were cotransfected with cDNAs encoding wild-type or mutant CACNB2b (Ca(v beta2b)), CACNA2D1 (Ca(v alpha2delta1)), and CACNA1C tagged with enhanced yellow fluorescent protein (Ca(v)1.2). Whole-cell patch-clamp studies were performed after 48 to 72 hours. Three probands displaying ST-segment elevation and corrected QT intervals < or = 360 ms had mutations in genes encoding the cardiac L-type calcium channel. Corrected QT ranged from 330 to 370 ms among probands and clinically affected family members. Rate adaptation of QT interval was reduced. Quinidine normalized the QT interval and prevented stimulation-induced ventricular tachycardia. Genetic and heterologous expression studies revealed loss-of-function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the alpha1- and beta2b-subunits of the L-type calcium channel. Confocal microscopy revealed a defect in trafficking of A39V Ca(v)1.2 channels but normal trafficking of channels containing G490R Ca(v)1.2 or S481L Ca(v beta2b)-subunits. This is the first report of loss-of-function mutations in genes encoding the cardiac L-type calcium channel to be associated with a familial sudden cardiac death syndrome in which a Brugada syndrome phenotype is combined with shorter-than-normal QT intervals.

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca V 1.2 channels during β1-adrenergic regulation

During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca(V)1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of beta-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the alpha(1) subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to beta-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective beta1- and beta2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only beta1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to beta1-adrenergic receptors. Thus, beta-adrenergic stimulation results in phosphorylation of S1928 on the Ca(V)1.2 alpha1 subunit in intact ventricular myocytes via both beta1- and beta2-adrenergic receptor pathways, but the beta2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with beta1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.

Redox regulation of cardiac calcium channels and transporters

Intracellular concentrations of redox-active molecules can significantly increase in the heart as a result of activation of specific signal transduction pathways or the development of certain pathophysiological conditions. Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca(2+) levels ([Ca(2+)](i)) and [Ca(2+)](i) regulation, redox modification of Ca(2+) channels and transporters has a profound effect on cardiac function. The sarcoplasmic reticulum (SR) Ca(2+) release channel, or ryanodine receptor (RyR), is one of the well-characterized redox-sensitive ion channels in the heart. The redox modulation of RyR activity is mediated by the redox modification of sulfhydryl groups of cysteine residues. Other key components of cardiac excitation-contraction (e-c) coupling such as the SR Ca(2+) ATPase and L-type Ca(2+) channel are subject to redox modulation. Redox-mediated alteration of the activity of ion channels and pumps is directly involved in cardiac pathologies such as ischemia-reperfusion injury. Significant bursts of reactive oxygen species (ROS) generation occur during reperfusion of the ischemic heart, and changes in the activity of the major components of [Ca(2+)](i) regulation, such as RyR, Na(+)-Ca(2+) exchange and Ca(2+) ATPases, are likely to play an important role in ischemia-related Ca(2+) overload. This article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of this redox regulation to normal and pathological cardiac function.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Calcium Current in Rat Cardiomyocytes Is Modulated by the Carboxyl-terminal Ahnak Domain

Ahnak, a protein of 5643 amino acids, interacts with the regulatory beta-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the beta(2)-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 +/- 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-beta(2)-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the beta(2)-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the alpha(1C)/beta(2)-subunit interaction and that the ahnak-derived proteins relieve the beta(2)-subunit from this inhibition.

Syntheses, calcium channel agonist-antagonist modulation activities, and nitric oxide release studies of nitrooxyalkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate racemates, enantiomers, and diastereomers.

A novel group of hybrid calcium channel (CC) modulators was prepared where the isopropyl ester moiety of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate (PN 202-791) was replaced by a variety of nitric oxide (*NO) donor nitrooxyalkyl ester substituents. Enantiomers, or diastereomers, having the (R)-configuration at the C-4 position of the 1,4-dihydropyridine ring (1,4-DHP) exhibited more potent in vitro CC antagonist activity on guinea pig ileum longitudinal smooth muscle (GPILSM) than compounds having the (4S)-configuration. None of the nitrooxyalkyl compounds exhibited a contraindicated CC agonist effect on GPILSM that would cause smooth muscle contraction. Structure-activity studies showed the enantiomers having the (S)-configuration at the C-4 position of the 1,4-DHP ring or diastereomers having the a (4S)-configuration at the C-4 position of the 1,4-DHP ring in conjunction with a (1R-)-1-methyl-2-nitrooxyethyl ester substituent exhibited the most potent cardiac CC agonist (positive inotropic) activity on guinea pig left atrium (GPLA). This class of compounds releases *NO in vitro that is enhanced by the presence of a thiol such as N-acetylcysteamine. The novel *NO donor (-)-(S,R)-1-methyl-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate [(-)-(S,R)-38], which acts as a dual cardioselective calcium channel agonist (GPLA)/smooth muscle selective calciumchannel antagonist (GPILSM), is a useful lead compound for drug discovery targeted to the treatment of congestive heart failure, and it provides a useful research probe to study the structure-function relationship of calcium channels.

Syntheses, calcium channel modulation effects, and nitric oxide release studies of O2‐alkyl‐1‐(pyrrolidin‐1‐yl) diazen‐1‐ium‐1,2‐diolate 4‐aryl(heteroaryl)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates

AbstractA group of racemic 4‐aryl(heteroary)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxy‐lates possessing a potential nitric oxide donor C‐5 O2‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester [alkyl=(CH2)n, n=1–4] substituent were synthesized using a modified Hantzsch reaction. Compounds having a C‐4 2‐trifluoromethylphenyl (16), 2‐pyridyl (17), or benzofurazan‐4‐yl (20) substituent generally exhibited more potent smooth‐muscle calcium channel antagonist activity (IC50 values in the 0.55 to 38.6 μM range) than related analogs having a C‐4 3‐pyridyl (18), or 4‐pyridyl (19) substituent with IC50 values &gt; 29.91 μM, relative to the reference drug nifedipine (IC50=0.0143 μM). The point of attachment of C‐4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2‐pyridyl &gt; 3‐pyridyl and 4‐pyridyl. Subgroups of compounds 16a–d, 17a–d, and 20a–d having alkyl spacer groups of variable chain length [–CO2(CH2)nO–, n=1–4] exhibited small differences in calcium channel antagonist potency. Replacement of the ester “methyl” moiety of Bay K 8644 by an O2‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate group provided the Bay K 8644 group of analogs 16a‐d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O2‐ethyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)pyridine‐5‐carboxylate (16b, EC50=0.096 μM) is about eightfold more potent positive inotrope (cardiac calcium channel agonist) than the reference compound Bay K 8644 (EC50=0.77 μM). A similar replacement of the ester “isopropyl” group in the C‐4 benzofurazan‐4‐yl group of compounds by an O2‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester substituent provided compounds 20 (n=1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202‐791 (3, EC50=9.40 μM). The O2‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester moiety present in 1,4‐dihydropyridine calcium channel modulating compounds 16–20 is not a suitable •NO donor moiety because the percent nitric oxide released upon in vitro incubation with either l‐cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204–216, 2003. © 2003 Wiley‐Liss, Inc.

Differential effects of fluoxetine enantiomers in mammalian neural and cardiac tissues

Racemic fluoxetine is a widely used SSRI antidepressant compound having also anticonvulsant effect. In addition, it was shown that it blocked several types of voltage gated ion channels including neural and cardiac calcium channels. In the present study the effects of enantiomers of fluoxetine (R(-)-fluoxetine and S(+)-fluoxetine) were compared on neuronal and cardiac voltage-gated Ca2+ channels using the whole cell configuration of patch clamp techniques, and the anticonvulsant action of these enantiomers was also evaluated in a mouse epilepsy model. In isolated pyramidal neurons of the dorsal cochlear nucleus of the rat the effect of fluoxetine (S(+), R(-) and racemic) was studied on the Ca2+ channels by measuring peak Ba2+ current during ramp depolarizations. All forms of fluoxetine reduced the Ba2+ current of the pyramidal cells in a concentration-dependent manner, with a Kd value of 22.3+/-3.6 microM for racemic fluoxetine. This value of Kd was higher by one order of magnitude than found in cardiac myocytes with fluoxetine enantiomers (2.4+/-0.1 and 2.8+/-0.2 microM). Difference between the effects of the two enantiomers on neuronal Ba2+ current was observed only at 5 microM concentration: R(-)-fluoxetine inhibited 28+/-3% of the peak current, while S(+)-fluoxetine reduced the current by 18+/-2% (n=13, P<0.05). In voltage clamped canine ventricular cardiomyocytes both enantiomers of fluoxetine caused a reversible concentration-dependent block of the peak Ca2+ current measured at 0 mV. Significant differences between the two enantiomers in this blocking effect was observed at low concentrations only: S(+)-fluoxetine caused a higher degree of block than R(-)-fluoxetine (56.3+/-2.2% versus 49.1+/-2.2% and 95.5+/-0.9% versus 84.5+/-3.1% block with 3 and 10 microM S(+) and R(-)-fluoxetine, respectively, P<0.05, n=5). Studied in current clamp mode, micromolar concentrations of fluoxetine shortened action potential duration of isolated ventricular cells, while higher concentrations also suppressed maximum velocity of depolarization and action potential amplitude. This shortening effect was significantly greater in the case of S(+) than R(-)-fluoxetine at 1 and 3 microM concentrations, whereas no differences in their effects on depolarization were observed. In pentylenetetrazole-induced mouse epilepsy model fluoxetine pretreatment significantly increased the 60 min survival rate, survival duration and seizure latency. These effects were more pronounced with the R(-) than the S(+) enantiomer. The results indicate that fluoxetine exerts much stronger suppressive effect on cardiac than neuronal calcium channels. At micromolar concentrations (between 1 and 10 microM) R(-)-fluoxetine is more effective than the S(+) enantiomer on neuronal, while less effective on cardiac calcium channels. The stronger anticonvulsant effect of the R(-) enantiomer may, at least partially, be explained by these differences. Used as an antidepressant or anticonvulsant drug, less severe cardiac side-effects are anticipated with the R(-) enantiomer.

Modulation of cardiac Ca(V)1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop.

Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of SerThr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca(V)1.2 (alpha(1)C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca(V)1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the alpha(1)C subunit or mimicking phosphorylation at that site.

A new promoter for α 1C subunit of human L-type cardiac calcium channel Ca V1.2

The cardiac Ca channel known as α 1C or Ca V1.2 is shown to express a new longer first exon equivalent to that formerly reported in rabbit heart or rat aorta. Ribonuclease protection assay indicates that this exon is found in the majority of Ca V1.2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven by a promoter immediately upstream of this exon and its 5 ′ untranslated region. The putative promoter exhibits 69% homology to its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs.

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation: USE-DEPENDENT BLOCK BY BENZOTHIAZEPINES

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. Single channel properties of mutants that lost use dependence toward diltiazem were characterized by drastically elongated mean open times and distinctly slower time constants of open time distribution. Mutation of individual residues of the IVMLF segment in IVS5 did not mimic the complete loss of use dependence as observed for the replacement of the whole stretch. These results establish evidence that amino acids that govern inactivation and the drug-binding site and other amino acids that are located distal from the putative drug-binding site contribute significantly to the function of the benzothiazepine receptor region. The data are consistent with a complex "pocket" conformation that is responsive to a specific class of L-type calcium channel inhibitors. The data allow for a concept that multiple sites within regions of the alpha(1) subunit contribute to auto-regulation of the L-type Ca(2+) channel.

Increased open probability of single cardiac L-type calcium channels of patients with chronic atrial fibrillation: role of phosphatase 2A

Increased open probability of single cardiac L-type calcium channels of patients with chronic atrial fibrillation: role of phosphatase 2A

Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.